The emergence of pancreatic islet transplantation as a means of restoring euglycemia represents a major milestone in the treatment of diabetes. However, the shortage of human donors underscores the importance of developing strategies to proliferate islets in vitro in order to increase the number of islet recipients per donor organ from starting islet preparations. There is evidence in rodents that mature beta cells retain a replicative potential although proliferating human beta cells have not been identified to date (Dor et al., (2004) Nature 429 (6897): 41-46). A major thrust in the study of islet proliferation has been the identification of endocrine progenitors that could potentially be expanded exponentially and then driven to express mature endocrine markers, in particular insulin. However, the identity of such progenitors remains to be elucidated.
The loss of islets during the isolation and cell culture process, as well as the loss of functionality in the grafts, is a significant hurdle in achieving sufficient numbers of islets to treat diabetic individuals. Human pancreatic cells cultured in vitro undergo apoptosis leading to a critical loss of endocrine cells. Establishing methods to preserve this material during the various steps prior to transplantation is therefore of critical importance.
During the process of organ procurement and cell isolation, islets are subject to multiple damaging insults resulting from donor brain death, organ isolation and preservation procedures, enzymatic digestion of the pancreas, isolation of the islet fraction, and in vitro cell culture; hence, the requirement for more than one donor pancreas to transplant a single patient. The activation of cell death mechanisms during all these steps is likely to explain the major reduction in functional islet mass observed before and after islet transplantation. This is a substantial constraint limiting the effectiveness of a procedure for the treatment of diabetes.
It is well established that caspases play a major role in the execution of various steps leading to apoptotic cell death. (Chandra, et al., (2001) Diabetes 50(supp 1):S44-S47). Inhibition of apoptosis has been shown to contribute to the successful expansion of endocrine cells from the limited number of human pancreas donors available for propagation. (See, e.g., U.S. Pat. No. 6,562,620; Hayek, A. et al., (2002) Curr. Diab Rep. 2:371-376). In particular, one approach has been to block the relevant cell death pathways responsible for islet loss and thus enhance the starting islet mass to be transplanted into the patient as disclosed in PCT Intl. Pub. No. WO 200361551. Although inhibition of one or more caspase members can prevent cell death in many cell types, including pancreatic endocrine cells, targeting cell death upstream of caspase cleavage may be more effective.
Several growth factors have been shown to enhance the survival, proliferation and function of islet cells. (Garcia-Ocaña, et al., (2001) J. Clin. Endocrinol. Metab. 86:984-988). These growth factors all act upstream of the phosphatidylinositol-3 kinase (P13K) signaling pathway which regulates the survival of various cell types (Stokoe, D. (2005) Expert Rev. Mol. Med. 7:1-22; Lin, et al., (1999) Cancer Research 59:2891-2897). A key effector of this pathway is the serine/threonine kinase, Akt, which is activated through membrane recruitment and phosphorylation. A role of Akt in the insulin response and in glucose metabolism of various tissues is well documented (Whiteman E. L., et al., (2002) Trends Endocrinol. Metab. 10:444-451). For example, mice harboring a deletion of the Akt2 gene exhibit growth deficiencies and are insulin resistant and glucose intolerant. A transgenic mouse expressing constitutively active Akt1 reportedly has both increased beta cell size and total islet mass leading to improved glucose tolerance and complete resistance to experimental diabetes (Tuttle, et al., (2001) Nat. Med. 7:1133-1137). Furthermore, recent studies have suggested that constitutive expression of Akt1 by either viral gene transfer or pharmacologic methods improves human islet transplant in diabetic mice (Contreras et al., (2001) Transplantation 74:1063-1069; Rao, et al., (2005) Diabetes 54:1664-1675).
Ultimately, the goal is to increase islet mass by blocking certain death pathways while simultaneously boosting proliferation and expansion of endocrine cells in order to make islet transplantation a practical approach to treating diabetes. The current invention achieves this goal by providing methods for improving the survival and cell proliferation of pancreatic endocrine cells in a pancreatic cell culture by contacting the cells with, (1) an exogenous caspase inhibitor in an amount sufficient to reduce apoptosis in the cells; and, (2) at least one exogenous growth factor in an amount sufficient to increase the level of activated Akt in the pancreatic endocrine cells. Moreover, the combination of the caspase inhibitors and growth factors provides a synergistic effect not seen in other cell types.